WO2020130626A1 - Aptamère d'adn se liant spécifiquement à ediii et utilisation correspondante - Google Patents
Aptamère d'adn se liant spécifiquement à ediii et utilisation correspondante Download PDFInfo
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- WO2020130626A1 WO2020130626A1 PCT/KR2019/017998 KR2019017998W WO2020130626A1 WO 2020130626 A1 WO2020130626 A1 WO 2020130626A1 KR 2019017998 W KR2019017998 W KR 2019017998W WO 2020130626 A1 WO2020130626 A1 WO 2020130626A1
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- ediii
- dna aptamer
- dengue virus
- aptamer
- dengue
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
- G01N2333/185—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a DNA aptamer specifically binding to dengue virus EDIII (Dengue virus envelope protein domain III; DENV EDIII), a composition for diagnosis of dengue fever comprising the same, and a dengue diagnosis kit.
- dengue virus EDIII Dengue virus envelope protein domain III
- DENV EDIII Dengue virus envelope protein domain III
- Dengue fever is a severe flu-like condition.
- the dengue virus belongs to a flavivirus, such as the yellow fever virus, and shows structural and pathological similarities. Symptoms include fever, severe headache, muscle and joint pain, and lymph node inflammation. Dengue fever itself has a low mortality rate, but there is a risk of complications such as hypothermia or hemorrhagic vomiting.
- dengue virus EDIII (Dengue virus envelope protein domain III; DENV EDIII) is a domain III part located on the surface protein of dengue virus, and the flavivirus to which the dengue virus belongs is inside the capsid protein.
- Genomic RNA genomic RNA
- E envelope
- M membrane protein
- the envelope protein located on the outermost side of the flavivirus antigen is composed of domains I, II, and III, of which domain III is a location where viral receptors bind, and has attracted attention as a major target for diagnosis and immune research using serum. have.
- Domain III is highly antigenic and has a linear epitope recognized by a virus-neutralizing single antibody. Diseases of the same flavivirus have a strong antigen-cross reaction between viruses, making it difficult for serological diagnosis. For example, there are reports that most infections have been misdiagnosed by a cross reaction between type 3 dengue fever, yellow fever, and St. Louis encephalitis. In order to solve this problem, the sequence of domain III having distinct characteristics even in viruses of the same genus is receiving attention (Registration No. 10-1520084, Republic of Korea).
- aptamer has a very high affinity for a specific material and is stable, and has been developed a lot in recent years, and its application as a therapeutic and diagnostic sensor using it is actively progressing. Synthesis of aptamers is possible in a relatively simple way, and even cells, proteins, and even small organic substances can be targeted, enabling the development of new detection methods using them, and their specificity and stability are very high compared to antibodies already developed. Therefore, it is possible to develop therapeutic agents, drug delivery systems, and applications as diagnostic biosensors.
- the present invention was devised to solve the above problems, and the specific binding capacity of the DNA aptamer prepared in the present invention to the dengue virus envelope protein domain III (DENV EDIII) was confirmed and based on this The invention was completed.
- the object of the present invention is a DNA aptamer that specifically binds to the Dengue virus EDIII (Dengue virus envelope protein domain III; DENV EDIII), wherein the DNA aptamer comprises a nucleotide sequence of SEQ ID NO: 4, DNA To provide aptamers.
- DENV EDIII Dengue virus envelope protein domain III
- another object of the present invention is to provide a dengue fever diagnostic kit or a composition for dengue fever diagnosis, which includes a DNA aptamer.
- the present invention is a DNA aptamer specifically binding to dengue virus EDIII (Dengue virus envelope protein domain III; DENV EDIII), wherein the DNA aptamer is the base sequence of SEQ ID NO: 4 It provides a DNA aptamer, characterized in that consisting of.
- DENV EDIII Dengue virus envelope protein domain III
- the DENV EDIII may be a domain protein included on the surface of the dengue virus antigen.
- the present invention provides a composition for diagnosing dengue fever, which includes a DNA aptamer.
- the present invention provides a dengue fever diagnostic kit comprising a DNA aptamer.
- the present inventors have conducted extensive research to develop a novel biomarker for the diagnosis of Dengue fever, and DNA aptamer (DNA) with specific binding ability to Dengue virus envelope protein domain III (DENV EDIII) Aptamer) has been confirmed to have strong binding strength and excellent specificity with DENV EDIII, and thus it can be expected to have superior stability than the ELISA method using an existing antibody, and thus can be usefully used in the development of dengue fever diagnostic composition, dengue fever diagnostic kit, etc. It is expected to be.
- DNA DNA aptamer
- DENV EDIII Dengue virus envelope protein domain III
- Figure 1 is a graph showing the results of confirming the binding degree (%) of ssDNA (single strand DNA) and DENV EDIII, and the screening process.
- Figure 2 shows the result of confirming the Kd value for the DV4 aptamer sequence through fluorescence measurement.
- Figure 3 shows the results of confirming whether the binding of DV1, DV2, DV3, DV4 aptamer with DENV EDIII protein through EMSA (Electrophoric Mobility Shift Assay) method.
- the present inventors have conducted extensive research to develop a novel biomarker for the diagnosis of Dengue fever, and DNA aptamer (DNA) with specific binding ability to Dengue virus envelope protein domain III (DENV EDIII) Aptamer) confirmed that it has a strong binding force with DENV EDIII and excellent specificity, and completed the present invention.
- DNA DNA aptamer
- DENV EDIII Dengue virus envelope protein domain III
- the present invention provides a DNA aptamer that specifically binds to DENV EDIII
- Dengue virus EDIII (Dengue virus envelope protein domain III; DENV EDIII)
- DENV EDIII domain III portion located on the surface protein of the Dengue virus, the Flavivirus to which the Dengue virus belongs
- Genomic RNA is located inside the capsid protein, and the outer surface has a structure surrounded by dimers and membrane proteins (M) proteins.
- M dimers and membrane proteins
- the envelope protein located on the outermost side of the flavivirus antigen is composed of domains I, II, and III, of which domain III is a location where viral receptors bind, and is receiving attention as a major target for diagnosis and immune research using serum. have.
- Domain III is highly antigenic and has a linear epitope recognized by a virus-neutralizing single antibody.
- aptamer is a single-stranded DNA (ssDNA) or RNA having high specificity and affinity for a specific substance.
- ssDNA single-stranded DNA
- RNA RNA having high specificity and affinity for a specific substance.
- Conventional methods using antibodies are relatively time-consuming and expensive because they are made using the immune system of a living body, and their stability may be problematic because they are proteins, whereas aptamers are relatively synthetic in synthesis. Since it is possible by a simple method and can target cells, proteins, and even small organic substances, it is possible to develop new detection methods using this, and the specificity and stability are very high compared to the antibodies already developed.
- DNA aptamers were used for the specific detection of EDIII. Any DNA (ssDNA) or RNA capable of specific detection of DENV EDIII may correspond to the aptamer of the present invention, and may be preferably composed of the nucleotide sequence of SEQ ID NO: 4, but is not limited thereto.
- the present invention provides a composition for diagnosing dengue fever, comprising the DNA aptamer.
- composition of the present invention may further include a pharmacologically or physiologically acceptable carrier, excipient, and diluent in addition to the DNA aptamer.
- a pharmacologically or physiologically acceptable carrier such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to a conventional method.
- Carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil.
- diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like.
- the present invention provides a dengue fever diagnostic kit comprising the DNA aptamer.
- the kit includes the aptamer of the present invention that specifically binds to DENV EDIII, and the aptamer may be attached with detection labels widely used in the art, for example, biotin residues.
- detection labels widely used in the art, for example, biotin residues.
- the labeling material After introducing the labeling material to the biotin-attached aptamer, it can be used for detection, quantification and diagnosis of DENV EDIII through its analysis.
- various known analytical labeling materials can be used, for example, fluorescence analysis using streptavidin, avidin, Cy3, Cy5, Alexa, BODIPY, Rhodamine or Q-dot, etc. Can.
- aptamers for DENV EDIII detection can be labeled with common labeling substances such as other fluorescent substances, magnetic substances, dyes, enzymes, radioactive isotopes, and common detection means, for example, fluorescence. It can be detected through a microscope, Radioimmnodetection (RAID), or the like.
- common labeling substances such as other fluorescent substances, magnetic substances, dyes, enzymes, radioactive isotopes, and common detection means, for example, fluorescence. It can be detected through a microscope, Radioimmnodetection (RAID), or the like.
- kit is a variety of tools or reagents that can be used to qualitatively or quantitatively determine whether aptamer is bound to DENV EDIII in a sample, a support (substrate), buffer solution, reaction stopper, solubilizer, detergent or stabilizer It may further include.
- the aptamer-fixed substrate may be, for example, a solid substrate, selected from the group consisting of polymers, glass, gold, paper, and biomembrane. More specifically, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethyl cellulose, polyacrylamide, Polysterene, companion rock, filter paper, ion exchange resin, plastic film, plastic tube, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, metal, glass, glass beads, or Magnetic particles and the like can be used.
- polystyrene polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethyl cellulose, polyacrylamide,
- Cell culture plates, ELISA plates, tubes, and polymeric membranes may be used as other solid substrates.
- the substrate may have any possible shape, for example spherical (bead), cylindrical (inside the test tube or well), planar (sheet, test strip), more preferably a multi-well plate (eg, 24 well) , 96 well, 192 well, 384 well, 576 well, etc.).
- the purified DENV EDIII was coated with cobalt on the surface and fixed to the bead using Dynabeads (Invitrogen, Norway), a magnetic bead that binds to the His-tag of the protein.
- the ssDNA library (500 pmole) dissolved in 100 uL of binding buffer was incubated at 90°C for 3 minutes, and then incubated at 4°C for 30 minutes. Then, it reacted for 1 hour while gently shaking with DENV EDIII previously immobilized on a magnetic bead. Then, the bead was washed twice with a binding buffer to remove ssDNA not binding to DENV EDIII bound to the bead.
- the protein and the elution buffer (20 mM Tris, 50 mM NaCl, 5 mM KCl, 5 mM MgCl 2 , 0.01% tween 20, 300 mM immidazole, pH 8.0) to separate the protein and ssDNA from the magnetic bead and The bound ssDNA was eluted.
- the eluted ssDNA was precipitated using ethanol, dissolved in 60 uL of distilled water, and then amplified by PCR using i-pfu polymerase (Intron Biotechnology, Korea) using Forward primer and Biotin-bound Reverse primer.
- the amount of ssDNA and the concentration of DENV EDIII were carried out while decreasing for strict screening, and the DENV of the remaining ssDNA was measured by measuring the concentration of ssDNA eluted from repeated screening with a UV spectrometer (UV spectrometer, Biochrom Libra S22 spectromether). The screening process was performed while confirming the degree of binding with EDIII. The degree of bonding (%) is as shown in FIG. 1.
- the 11th repeatedly selected ssDNA was amplified through PCR using unmodified Forward primer and Reverse primer, cloned into pENTR/TOPO vector (TOPO TA Cloning kit, Invitrogen, USA), and E. coli TOP10 cells (Invitrogen, USA). After the clone into which the ssDNA was inserted was purified using a miniprep kit (GeneAll, Korea), the base sequence was analyzed (COSMO Genetech, Korea). As a result, it was possible to analyze the sequence shown in Table 1 below.
- the amount of 6-FAM-labeled ssDNA aptamer bound to magnetic beads with DENV EDIII fixed was separated by fluorescence measurement by separating only 6-FAM-labeled ssDNA bound to DENV EDIII using a magnet (1420 Victor multilabel counter, PerkinElmer, USA).
- a magnet (1420 Victor multilabel counter, PerkinElmer, USA.
- Example 2 In order to measure whether the DV4 aptamer selected through the SELEX (Systematic Evolution of Ligands by Exponential enrichment) technique of Example 1 binds to the actual DENV EDIII, the present inventors confirm the presence or absence of binding through the EMSA (Electrophoric Mobility Shift Assay) method. Did. Recombinant DENV EDIII 5 uM and aptamers DV1, DV2, DV3, and DV4 1 uM were incubated in a binding buffer of 20 uL at room temperature for 1 hour, and then run on 6% acrylamide gel. As a result of sybr green and coomassie blue staining, as shown in FIG. 3, it was confirmed that the developed DV4 aptamer binds to DENV EDIII better than other aptamers.
- SELEX Systematic Evolution of Ligands by Exponential enrichment
- Sequence list designation order SEQ ID NO: 1 ssDNA library CACCTAATACGACTCACTATAGCGGATCCGA-N40-CTGGCTCGAACAAGCTTGC SEQ ID NO: 2 Forward primer cacctaatac gactcactat agcgga SEQ ID NO: 3 Reverse primer gcaagcttgt tcgagccag SEQ ID NO: 4 DV4 Aptamer CACCTAATACGACTCACTATAGCGGATACGAGGGAGAGTCGGATGGGGTGGTTGTGTTAGGTGTAGTGGGTCTGGCTCGAACAAGCTTGC
- the DNA aptamer that specifically binds to the dengue virus envelope protein domain III (DENV EDIII) of the present invention has a strong binding force and excellent specificity to the dengue virus surface protein, and is significantly remarkable compared to the diagnosis using existing antibodies. Since dengue virus can be diagnosed with increased accuracy, it is expected that it can be effectively used in various products for diagnosis, such as diagnostic kits and diagnostic compositions.
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Abstract
La présente invention concerne un aptamère d'ADN se liant spécifiquement au domaine III de la protéine d'enveloppe du virus de la dengue (DENV EDIII), une composition le comprenant pour diagnostiquer la dengue, et un kit pour le diagnostic de la dengue. La recherche Intensive et approfondie, réalisée par les présents inventeurs, dans le développement d'un nouveau biomarqueur pour Le diagnostic de la dengue a conduit à la découverte d'un aptamère d'ADN capable de se lier spécifiquement au domaine III de la protéine d'enveloppe du virus de la dengue (DENV EDIII) présentant une forte affinité de liaison et une excellente spécificité pour DENV EDIII. L'aptamère d'ADN est supposé être plus stable que les procédés ELISA classiques utilisant des anticorps et, en tant que tel, peut être avantageusement utilisé dans le développement d'une composition pour le diagnostic de la dengue et d'un kit pour le diagnostic de la dengue.
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KR20230107461A (ko) | 2022-01-08 | 2023-07-17 | 광운대학교 산학협력단 | 중증 뎅기열 면역진단기기를 이용한 중증 뎅기열 관제 시스템 및 이에 의한 중증 뎅기열 관제방법 |
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KR101520084B1 (ko) | 2014-01-09 | 2015-05-29 | 주식회사 젠바디 | 뎅기열 바이러스의 표피단백질 도메인1에 특이적인 단클론 항체를 포함하는 뎅기열 바이러스 항체의 신속진단키트 및 그 제조 방법 |
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